Cell Cultivations

The purpose of this chapter is to give art introduction to the basics of microbial, animal, and plant cells and their cultivation techniques and applications. It covers only what is necessary for understanding the terminologies and procedures introduced in this book.1 

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Table 3. Environmental Parameters for Mammalian Cell Cultivation...

Fed-batch culture A cell cultivation technique in which one or more nutrients are supplied to the bioreactor in a given sequence during the growth or bioconversion process while the products remain in the vessel until the end of the run. 

Where yield coefficients are constant for a particular cell cultivation system, knowledge of how one variable changes can be used to determine changes in the other. Such stoichiometric relationships can be useful in monitoring fermentations. For example, some product concentrations, such as CO2 leaving an aerobic bioreactor, are often the most convenient to measure in practice and give information on substrate consumption rates, biomass formation rates and product formation rates. 

If you were unable to do these calculations, we would suggest that you read the BIOTOL text entitled In vitro Cultivation of Micro-oiganisms or other suitable texts of the fundamental principles of cell cultivation. 

The culture can be used directly for the conversion of phenylpyruvic add to resting cells L-phenylalanine. Therefore, a batch process with resting cells can be carried out, with some glucose added for maintenance (fed-batch fermentation). Another approach is to harvest the cells from the fermentation broth and to use them in a separate bioreactor in higher concentrations than the ones obtained in the cell cultivation. An advantage of the last method can be that the concentration of compounds other than L-phenylalanine is lower, so that downstream processing may be cheaper. 

S. J. Pirt, Principles of Microbe and Cell Cultivation, Blackwell, Oxford (1975). J. R. Postgate, New advances and future potential in biological nitrogen fixation. Journal of Applied Bacteriology 57 185 (1974). 

N., Sjostrom, B., Automated absorption assessment using Caco-2 cells cultivated on both sides of polycarbonate membranes, Pharm. 

A continuous centrifugal bioreactor, in which cells are fluidized in balance with centrifugal forces, has been designed to allow high density cell cultivation and superior aeration without elutriation of the suspended cells (van Wie et al., 1991). Reactor performance was hampered by elutriation of biomass by evolved gas in an anaerobic fermentation, indicating that it may not be suitable in its present state for three-phase fermentations. Immobilization of the cells on denser particles may overcome this problem. 

Funfak A, Cao J, Wolfbeis OS, Martin K, Kohler JM (2009) Monitoring cell cultivation in microfluidic segments by optical pH sensing with a micro flow-through fluorometer using dye-doped polymer particles. Microchim Acta 164 279-286... 

R. rubrum was shown to possess hydrogenase and its activity was followed for 72 h. The bacteria were grown synchronously under anoxic conditions, with N2 or CO2 gas, at 30°C and in absence or presence of SeO . The production of dimethyl selenide and dimethyl diselenide was detected by chemiluminescence detection of samples of the gas phase (Fig. 9.12). After a 24 h exposure to complete darkness, the bacteria were transferred to the experimental conditions and changes in the activity of hydrogenase with time were studied in non-growing cells, cultivated in white light. 

The first batch of cells consisted of AC 133+ cells cultivated in the diffusion chambers submerged on top of the feeder (feeder -AC 133 cells /Fl-C). The second batch consisted of human embryonic liver cell suspension directly cocultured with AC133+ cells at equal initial quantities (5x10 ) in the diffusion chambers submerged in the 6-well plates without additional feeder layer (FC-C). Third batch of experiments represented AC133+ cells cultured in the DC surrounded by FL condition media (condition media-AC133+ cells/CM-C). In the control group cells were cultivated in the same condition without any additions and without feeder layers. 

Santos, J.C., Silva, S.S., Mussatto, S.I., Carvalho, W. and Cunha, M.A.A., Immobilized cells cultivated in semi-continuous mode in a fluidized bed reactor for xylitol production from sugarcane bagasse, World. Microbiol. Biotech., 21 (2005) 531-535. 

Courtois, D. and Guern, J. 1980. Temperature response of Catharanthus roseus cells cultivated in liquid medium. Plant Science Letters, 17 473 82. 

These optical probes are the most universally applicable in situ devices for on-line biomass monitoring up to now [15,16]. Konstaninov et al. [17] tested several absorbance and scattering sensors for real-time biomass concentration monitoring in mammalian cell cultivation processes and Hatch and Veilleux [18] compared optical density probes with oxygen uptake rates, packed cell volume, and off-line cell mass monitoring in commercial fed-batch fermentations of Saccharomyces cerevisiae [19]. In order to minimize influencing effects, special chemometric data treatment is necessary [20]. 

In order to quantify the physical environment of a bioreactor, fluorescence assays can be applied for on-line monitoring of the mixing time behavior of all types of bioreactors. In this case the fluorosensor probe can be installed in the bioreactor at different locations of interest. Afterwards, selected fluorophores can be injected in order to study the overall mixing time. These fluorophores must fit to the excitation and emission behavior of the probe and should be selected in regard to the pH-dependency of the bioprocess, and when used during cell cultivation experiments they should not interfere with the cells. Scheper and Schiigerl reported on the use of different coumarins for mixing time experiments under bioprocess relevant conditions [49]. 

Due to the complexity and the manifold interactions between cytokines, determination of an optimum cytokine mix for stem cell cultivation is difficult and most optimization algorithms are inefficient. Significant progress can be made using genetic algorithms, as shown by Thoma et al. [41]. 

Three different approaches for the cultivation of isolated hematopoietic cells have been described, the static, the stirred and the immobilized culture. Static cultivation takes place in very simple culture systems like well plates, tissue-culture flasks or gas-permeable culture bags [62, 63]. As the first two systems do not allow cell cultivation on a clinical scale, the latter is actually the most often used technique for stem cell expansion. All these systems have the advantage of being easy to handle, single-use devices, which enable an uncomplicated cell harvest. But all of them do not offer possibilities for process control or continuous feeding. This causes variations in culture conditions during fermentation (e.g., oxygen tension, pH, substrate, metabolite and cytokine concentrations). 

Figure 4.11. a so liter bench fermenter that can be scaled for production of recombinant proteins. The bench-top scale configuration contains all the control valves and ports necessary to monitor and control cell cultivation while maintaining sterility of the culture. The stainless steal reaction vessel allows easy cleaning and permits heat and pressure sterilization in place by connecting the vessel to a steam supply. (New Brunswick Bioflo-4500, adapted from the manufacturer s literature with permission)... 

Figure 4.12. Configuration of large-scale, cell cultivation methods for pharmaceutical scale production...

Fermentation and alternative production techniques, such as roller bottles, can be carried out in four different ways. They are (1) batch process, (2) fed-batch process, (3) chemostat process, and (4) perfusion process. Batch and fed-batch processes require termination of cell growth while chemostat and perfusion processes allow continuous cell cultivation. 

Figure 4.13. Schematic presentation of process stream to purify a recombinant protein, starting from cell suspension harvested from fermenter or cell-cultivation vessels.

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